猪瘟病毒血清抗体间接ELISA检测方法的建立  

Development of an indirect ELISA for the detection of antibody against classical swine fever virus

作　　者：蔺辉星[1,2] 周红[1] 童泽鑫 范红结[1,2] 

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]江苏高校动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《中国兽医科学》2017年第1期9-15,共7页Chinese Veterinary Science

基　　金：江苏省农业科技自主创新资金项目(CX(15)1056;CX(16)1028)

摘　　要：为建立快速、准确的猪瘟病毒(CSFV)血清抗体检测方法,根据Gen Bank上猪瘟兔化弱毒全基因序列,设计1对引物,扩增E2蛋白主要抗原区基因片段;然后,将其克隆入载体p ET-28a(+),采用大肠杆菌Rosetta(DE3)表达出含E2蛋白主要抗原区的重组蛋白。以纯化的截短E2蛋白为包被抗原,优化间接ELISA反应条件,建立了检测CSFV血清抗体的间接ELISA。经过优化,确定最佳抗原包被浓度为8 g/m L,待检血清稀释倍数为1∶160,酶标二抗稀释倍数为1∶2000。该方法的阳性临界值为0.37,阴性临界值为0.32。批内变异系数在1.58%~3.33%之间,批间变异系数在2.67%~5.35%之间,说明该方法重复性良好。特异性检验中,该方法与猪繁殖与呼吸综合征病毒、猪圆环病毒2型、猪流行性腹泻病毒、猪流感病毒、传染性胃肠炎病毒、乙型脑炎病毒、伪狂犬病病毒、猪细小病毒阳性血清均没有交叉反应。用该方法与IDEXX CSFV抗体检测试剂盒对1 101份临床猪血清样品进行符合性检测,结果,该方法的敏感性、特异性、符合率分别为89.07%(603/677)、77.59%(329/424)和84.65%(932/1101)。上述结果表明,该方法可用于临床CSFV血清抗体的检测,为CSFV血清抗体检测试剂盒的研制奠定了基础。In order to develop a rapid and accurate antibody test to detect antibodies against classical swine fever virus（CSFV）, the gene of main antigenic region of CSFV E2 protein was amplified. The RT-PCR product was cloned into pET-28a （＋） vector. Then the truncated E2 protein was expressed in coli system and this indirect ELISA diagnostic method was developed using this truncated E2 protein as a coating antigen. In the optimal process for the CSFV indirect ELISA,the truncated E2 protein was coated at 8 μg/mL,swine serum samples were diluted by 1：160 and rabbit anti-pig IgG-HRP was diluted by 1：2000. The positive and negative cut-off values of this assay were 0.37 and 0.32,respectively. The co- efficients of variation（CV） of inter-assay was between 1.58%and 3.33%,and the CV of intra-assay was between 2.67% and 5.35%.There were no cross-reactions with the positive sera of porcine reproduc- tive and respiratory syndrome virus,porcine circovirus type 2,porcine epidemic diarrhea virus,swine influenza virus,transmissible gastroenteritis virus,Japanese encephalitis virus,porcine pseudora- hies virus and porcine parvovirus. The assay was applied to examine 1101 swine serum samples. Compared with IDEXX CSFV ELISA kit,the sensitivity,specificity and coincidence rates were 89.07 %（603/677）, 77.59^（329/424） and 84.65^（932/l101）,respectively. The results indicated that the developed CSFV indirect ELISA would be a rapid and accurate method for monitoring antibodies against CSFV.

关 键 词：猪瘟病毒 E2蛋白主要抗原区 间接ELISA 

分 类 号：S852.651[农业科学—基础兽医学]